Inhibition of respiratory complex iii by ligands that interact with a regulatory switch

ABSTRACT

The present invention provides methods for inhibiting respiratory complex III in a cell. The present invention also provides methods for treating cancer in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. application Ser. No.16/531,511, filed Aug. 5, 2019, which is a continuation of InternationalPatent Application No. PCT/US2018/017638, filed Feb. 9, 2018, whichclaims priority to U.S. Provisional Appln. No. 62/457,684, filed Feb.10, 2017, the disclosures of which are herein incorporated by referencein their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. GM054052awarded by the National Institutes of Health (NIH). The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Ideally, a smart anti-cancer drug should discriminate between cancer andnormal cells. One of the recently discovered biochemical variations incancer cells that distinguish them from normal cells is a higher basallevel of reactive oxygen species (ROS), which makes the cancer cellsmore susceptible to ROS-induced apoptosis (Szatrowski and Nathan (1991)Cancer Res. 51:794; Kawanishi et al. (2006) Biol. Chem. 387:365;Toyokuni et al. (1995) FEBS Lett. 358:1; and Trachootham et al. (2009)Nat. Rev. Drug Discov. 8:579). However, since cancer cells can adapt tosuch oxidative stress by up-regulating antioxidant production (Tiligada(2006) Endocrine Related Cancer 13:S115), to make use of such amechanism, a drug should induce rapid production and accumulation of ROSand trigger apoptosis in cancer cells before anti-oxidant up-regulationtakes effect.

The mitochondrial electron transport chain (METC) is one of the majorsources of ROS in the cell (Adam-Vizi and Chinopoulos (2006) TrendsPharmacol. Sci. 27:639; and Lenaz (2001) IUBMB Life 52:159), andrespiratory complex III (also known as ubiquinol:cytochrome coxidoreductase complex or bc₁ complex) is one of the two chief producersof ROS in METC (Turrens and Boveris (1980) Biochem. 1191:421; andSugioka et al. (1988) Biochim. Biophys. Acta 936:377). Therefore, bc₁complex is a natural target for any candidate anti-cancer drug whosefunction would be to increase ROS production and to bring it to anover-threshold level and thereby trigger apoptosis in cancer cells,while leaving the normal cells in under-threshold level of ROS(Trachootham et al. (2009) Nat. Rev. Drug Discov. 8:579). Moreover, bc₁complex has a docking site for the water-soluble electron carriercyctochrome c which plays a critical role in cell apoptosis (Jiang andWang (2004) Annu. Rev. Biochem. 73:87). Thus, bc₁ complex has a dualrole in this context: as a major site for ROS production which exerts acytotoxic effect (Fruehauf and Myskens, Jr. (2007) Clin. Cancer Res.13:789), and as a source of oxidative stress leading to cytochromec-mediated cell apoptosis.

Located in the inner-mitochondrial membrane, bc₁ complex is the middleplayer in the electron transport proton pumping orchestra, whichconverts the free energy of redox reactions to an electrochemical protongradient (Mitchell (1961) Nature 191:141 and Mitchell (1966) Biol. Rev.Camb. Philos. Soc. 41:445). The mitochondrial bc₁ complex has anintertwined dimeric structure comprised of 11 subunits in each monomer,but only three of them have catalytic function. The core subunitsinclude: the Rieske domain, which incorporates an iron-sulfur cluster[2Fe-2S]; the trans-membrane cytochrome b domain, incorporating alow-potential heme group (heme b_(L)) and a high-potential heme group(heme b_(H)); and the cytochrome ci domain, containing heme ci group andtwo separate binding sites, Q_(o) (or Q_(P)) site where the hydrophobicelectron carrier ubihydroquinol QH₂ is oxidized, and Q_(i) (or Q_(N))site where ubiquinone molecule Q is reduced (Xia et al. (1997) Science277:60; Schagger et al. (1986) Methods Enzymol. 126:224; Zhang et al.(1998) Nature 392:677; and Iwata et al. (1998) Science 281:64).

Because of the relevance of respiratory complex III to ROS-inducedapoptosis of cancer cells, study of the enzyme has been an active fieldof research. U.S. Patent Application Publication No. 2005/0019766describes the characterization of mutants in the genes isp-1 and ctb-1,which are genes that have a function at the level of cellularphysiology, mitochondrial respiration and electron transport, andresistance to oxidative stress, as well as regulating developmental,behavioral, reproductive and aging rates. U.S. Patent ApplicationPublication No. 2008/0280294 describes a method for identifying asubject likely to have, or at risk of developing a disease conditioncorrelated with increased ROS, including cancer, by identifying in thesubject a missense mutation in a nucleic acid of complex III, IV and/orV of the oxidative phosphorylation system. U.S. Patent ApplicationPublication No. 2011/0144192 describes a method for modulating orcontrolling sodium channel current of a cell including inducingmitochondrial ROS production in the cell. U.S. Pat. No. 8,815,844describes inhibitors of the activity of the electron transport chainsand/or the mitochondrial TCA cycle in glioma-initiating cells (GICs) foruse in a method for preventing and/or treating tumors presentingglioma-initiating cells (GICs) in a subject who has undergone a priorremoval of a tumor glioma bulk.

Even in view of these references, the need exists for novel inhibitorsof the activity of the mitochondrial electron transport chain.Accordingly, in addition to fulfilling other needs, the presentinvention provides methods for inhibiting respiratory complex III. Assuch, the present invention provides methods that can be useful astherapies for disorders such as cancer in patients.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for using several ligandcompounds to bind to respiratory complex III and inhibit the functioningof the complex. A newly identified binding site is located at theopposite side of the respiratory complex III enzyme with respect to theubiquinol-binding site (Q_(o) site), and distinctly different from bothQ_(o) and Q_(i) sites (hence designated as Non-Q binding site, NQ).NQ-site binding pocket extends up close to Phe90 residue, an internalswitch (LH switch) that regulates electron transfer between heme b_(L)and heme b_(H) of the low potential redox chain. Docking studies andmolecular dynamics simulations of different molecules to NQ-siterevealed potential ligands which exhibit a novel inhibitory effect forbc₁ complex by switching the LH switch to an “off” conformation, therebysignificantly reducing electron transfer rate in the low potential redoxchain. Moreover, the novel inhibitors have lower binding affinity forboth Q_(o) and Q_(i) sites, and hence do not interfere with binding ofthe natural ligands to those sites. The inhibitory activity of thosenovel ligands in bc₁ complex can promote the production of reactiveoxygen species (ROS) at the Q_(o) site. Hence those ligands arepotential candidates for designing new “mitocan” drugs.

In one aspect, the present invention provides a method of inhibitingrespiratory complex III in a cell. The method includes contacting thecell with a compound such that the compound binds to respiratory complexIII, thereby inhibiting respiratory complex III. The compound can havethe structure:

Each R¹, R², R³, R⁴, R⁵, and R⁶ can independently be hydrogen, C₁₋₆alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and —C(O)—C₁₋₆alkyl, or —C(O)O⁻. The subscripts m and n can each independently beintegers from 0 to 5. The subscript p is an integer from 0 to 4. Thesubscripts q, u, and v can each independently be integers from 0 to 2.The subscript w is an integer from 0 to 3.

In some embodiments, the compound enhances the production of reactiveoxygen species (ROS) in the cell. In some embodiments, the cell is acancer cell. In some embodiments, the cell is in a subject. In someembodiments, the method further includes administering an effectiveamount of the compound to the subject.

In a further aspect, the present invention provides a method of treatingcancer in a subject. The method includes administering to the subject inneed thereof, a therapeutically effective amount of a compound that canhave the structure:

Each R¹, R², R³, R⁴, R⁵, and R⁶ can independently be hydrogen, C₁₋₆alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and —C(O)—C₁₋₆alkyl, and —C(O)O⁻. The subscripts m and n can each independently beintegers from 0 to 5. The subscript p is an integer from 0 to 4. Thesubscripts q, u, and v can each independently be integers from 0 to 2.The subscript w is an integer from 0 to 3.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of the homodimer respiratory complexIII embedded in the membrane where the chains of cytochrome b domain areeither removed or transparent for clarity. Relative positions are shownof heme b_(L) and heme b_(H) with the intervening internal switch Phe90residue. Different binding sites are visualized including Q_(o) bindingpocket as computed with AutoLigand occupied by QH₂ molecule (van derWaals spheres), Q molecule occupying Q_(i) site (van der Waals spheres)and NQ site as computed by AutoLigand (van der Waals spheres). theinset, zoomed-in visualization of NQ site entrance is displayed.

FIG. 2 presents different ligand binding motif chemical structures forNQ-site in respiratory complex III.

FIG. 3 is a graph of docking results of the preliminary ligand set atthe NQ-site using fixed residues against flexible residues. The positivedifference of the binding energies (fixed-flexible) is plotted at thetop. White lines represent the cut-off binding energy of QH₂ molecule atNQ site.

FIG. 4 is a graph of docking results at Q_(o) site (1NTZ in PDB) of theselected 30 ligands from FIG. 3 using Vina (light grey bars) and LG(dark grey bars) search algorithms. Black bars represent the overlapbetween Vina and LG corresponding bars.

FIG. 5 shows chemical structures of five candidate ligands.

FIG. 6 illustrates electron tunneling flux through a dividing surface inheme b_(L)→heme b_(H) redox system at three different Phe90conformations (ON_(LH), OFF_(LH) and OFF_(NQ)). The flux plot shows theedge-to-edge tunneling flux at the different conformations plotted aslog₁₀ of the flux (adjusted by multiplying by 10) against the normalizedcoordinate.

FIG. 7 illustrates docking of the selected ligands of FIG. 5 at NQ site.Different conformations of the flexible residues including the internalswitch Phe90 residue are shown. The arrows signify the change fromnative ON_(LH) conformation to the new ones.

FIG. 8 illustrates calculated electron tunneling flux densities betweenheme b_(L) and heme b_(H) in two X-ray bc₁ crystal structures (PDB 1NTZin A and C and 1NTK in B). Intensity of darkness corresponds toprobability of a tunneling electron being on a given atom. A. residuePhe90 exists in ON_(LH) conformation. B. residue Phe90 exists inOFF_(LH) conformation. C. residue Phe90 exits in OFF_(NQ) conformation.

FIGS. 9A-9C present molecular dynamics simulation results of the fivediscovered ligands of FIG. 5 docked in pre-energy minimized bc₁ complex(PDB: 1BE3). FIG. 9A: plots of the total distance between thecorresponding ligand centroid and both heme b iron atoms along the 50 nsMD trajectory. FIG. 9B: plots of the corresponding ligand RMSD along the50 ns MD trajectory. FIG. 9C: plots of the total distance between Phe90centroid and both heme b porphyrin ring edges along the 50 ns MDtrajectory. The baseline (=13.3 Å) equals to the distance found inQ-bound bc₁ structure (PDB: 1NTZ) (indicated by hashed line if totaldistance is lower than 13.3 Å).

FIG. 10 is a visualization of 1000 snapshots of two different dockedligands (top, ZINC01691943 and bottom, ZINC17147424) at NQ-site in bc₁structure (PDB: 1BE3) along with snapshots of the conformational changesof the Phe90 residue during the 50 ns MD run. The snapshots vary indarkness based on the simulation time-step.

FIG. 11 is a graph of Autodock Vina docking results of the ZINC databaseligands set at the NQ-site using fixed residues against flexibleresidues. The positive differences of the binding energies are plottedat the top as positive values.

FIG. 12 is a graph of Fix-Flex binding energies at NQ site for theselected 2,969 ligands using Vina and LG search algorithms.

FIG. 13 is a graph of docking results at Q_(o) site (1NTZ in PDB) of theselected 41 ligands from FIG. 12 using Vina (light grey bars) and LG(dark grey bars) search algorithms. Black bars represent the overlapbetween Vina and LG corresponding bars.

FIG. 14 shows chemical structures of representative ligands in fourclasses.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Residue Phe90 plays the role of an intra-monomeric switch (designated asLH switch) which can regulate the rate of electron transfer between hemeb_(L) and heme b_(H) of the bc₁ respiratory complex III (Hagras et al.(2016) Biochim. Biophys. Acta 1857:749). Phe90 can exist in twoconformations, designated as OFF_(LH) and ON_(LH). The binding of anaromatic ligand at Q_(o) site switches the conformation of Phe90 from atilted conformation (OFF_(LH)) to a parallel conformation (ON_(LH)),facilitating electron transfer reaction between heme b_(L) and hemeb_(H). Being in the OFF_(LH) conformation, the electron transfer ratediminishes by about three orders of magnitude compared to the Phe90ON_(LH) conformation. Modification of the conformation of the electrontransfer switch can interfere with the trans-membrane ET reactionbetween heme b_(L) and heme b_(H) to slow it down, or shut it offcompletely. Such interference can lead to a greater localization of thesecond electron (transferred from QH₂ at Q_(o) site) on heme b_(L) andhence to a higher chance to be picked up by oxygen molecules leading toROS production.

A unique binding pocket extends deep into the enzyme body reaching theregion of Phe90 switch. This new binding pocket (designated as NonQ-siteor NQ-site for short) in the respiratory complex III complex is shown inFIG. 1. The NQ site is located at the opposite side of the enzyme withrespect to Q_(o) site; like Q_(o), it is buried inside theinner-mitochondrial membrane, and its entrance is fully hydrated. Incontrast to Q_(o) site, however, the NQ-site penetrates deeply in thecytochrome b domain and reaches very closely the LH region. Hence theNQ-site provides a suitable binding pocket for ligands that caninfluence the orientation of Phe90 residue, and hence modulate thecorresponding ET rate between heme b_(L) and heme b_(H).

A series of docking calculations and molecular dynamics simulations atthe new docking site has led to a set of ligands that fix the Phe90switch to its OFF position and hence significantly diminish the electrontransfer rate between heme b_(L) and heme b_(H). The inhibiting ligandscan result in a major increase of ROS production by the enzyme, and thuscan be utilized to design a smart mitocan drug.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “administering” refers to oral administration, administrationas a suppository, topical contact, parenteral, intravenous,intraperitoneal, intramuscular, intralesional, intranasal orsubcutaneous administration, intrathecal administration, or theimplantation of a slow-release device e.g., a mini-osmotic pump, to thesubject.

The term “subject” refers to animals such as mammals, including, but notlimited to, primates (e.g., humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice and the like. In certain embodiments, thesubject is a human.

The terms “therapeutically effective amount or dose” or “therapeuticallysufficient amount or dose” or “effective or sufficient amount or dose”refer to a dose that produces therapeutic effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, thetherapeutically effective dose can often be lower than the conventionaltherapeutically effective dose for non-sensitized cells.

The term “alkyl” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated. Alkyl can includeany number of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋ ₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 20 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc. Alkyl groups can be substituted orunsubstituted.

The term “alkylhydroxy” refers to an alkyl group, as defined above,where at least one of the hydrogen atoms is replaced with a hydroxygroup. As for the alkyl group, alkylhydroxy groups can have any suitablenumber of carbon atoms, such as C₁₋₆. Exemplary alkylhydroxy groupsinclude, but are not limited to, hydroxy-methyl, hydroxyethyl (where thehydroxy is in the 1- or 2-position), hydroxypropyl (where the hydroxy isin the 1-, 2- or 3-position), hydroxybutyl (where the hydroxy is in the1-, 2-, 3- or 4-position), hydroxypentyl (where the hydroxy is in the1-, 2-, 3-, 4- or 5-position), hydroxyhexyl (where the hydroxy is in the1-, 2-, 3-, 4-, 5- or 6-position), 1,2-dihydroxyethyl, and the like.

The term “alkoxy” refers to an alkyl group having an oxygen atom thatconnects the alkyl group to the point of attachment: alkyl-O—. As foralkyl group, alkoxy groups can have any suitable number of carbon atoms,such as C₁₋₆. Alkoxy groups include, for example, methoxy, ethoxy,propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy,tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be furthersubstituted with a variety of substituents described within. Alkoxygroups can be substituted or unsubstituted.

The term “haloalkyl” refers to alkyl, as defined above, where some orall of the hydrogen atoms are replaced with halogen atoms. As for alkylgroup, haloalkyl groups can have any suitable number of carbon atoms,such as C₁₋₆. For example, haloalkyl includes trifluoromethyl,flouromethyl, etc. In some instances, the term “perfluoro” can be usedto define a compound or radical where all the hydrogens are replacedwith fluorine. For example, perfluoromethyl refers to1,1,1-trifluoromethyl.

The term “haloalkoxy” refers to an alkoxy group where some or all of thehydrogen atoms are substituted with halogen atoms. As for an alkylgroup, haloalkoxy groups can have any suitable number of carbon atoms,such as C₁₋₆. The alkoxy groups can be substituted with 1, 2, 3, or morehalogens. When all the hydrogens are replaced with a halogen, forexample by fluorine, the compounds are per-substituted, for example,perfluorinated. Haloalkoxy includes, but is not limited to,trifluoromethoxy, 2,2,2,-trifluoroethoxy, perfluoroethoxy, etc.

The term “aryl” refers to an aromatic ring system having any suitablenumber of ring atoms and any suitable number of rings. Aryl groups caninclude any suitable number of ring atoms, such as, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6to 14 ring members. Aryl groups can be monocyclic, fused to formbicyclic or tricyclic groups, or linked by a bond to form a biarylgroup. Representative aryl groups include phenyl, naphthyl and biphenyl.Other aryl groups include benzyl, having a methylene linking group. Somearyl groups have from 6 to 12 ring members, such as phenyl, naphthyl orbiphenyl. Other aryl groups have from 6 to 10 ring members, such asphenyl or naphthyl. Some other aryl groups have 6 ring members, such asphenyl. Aryl groups can be substituted or unsubstituted.

The term “halogen” refers to fluorine, chlorine, bromine and iodine.

The term “cancer” is intended to include any member of a class ofdiseases characterized by the uncontrolled growth of aberrant cells. Theterm includes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,breast cancer; lung cancer (e.g., non-small cell lung cancer); digestiveand gastrointestinal cancers such as colorectal cancer, gastrointestinalstromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectalcancer, anal cancer, bile duct cancer, small intestine cancer, andstomach (gastric) cancer; esophageal cancer; gallbladder cancer; livercancer; pancreatic cancer; appendix cancer; ovarian cancer; renal cancer(e.g., renal cell carcinoma); cancer of the central nervous system; skincancer; lymphomas; choriocarcinomas; head and neck cancers; osteogenicsarcomas; and blood cancers. As used herein, a “tumor” comprises one ormore cancerous cells.

“Salt” refers to acid or base salts of the compounds used in the methodsof the present invention. Illustrative examples of pharmaceuticallyacceptable salts are mineral acid (hydrochloric acid, hydrobromic acid,phosphoric acid, and the like) salts, organic acid (acetic acid,propionic acid, glutamic acid, citric acid and the like) salts,quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.It is understood that the pharmaceutically acceptable salts arenon-toxic. Additional information on suitable pharmaceuticallyacceptable salts can be found in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., 1985, which isincorporated herein by reference.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts.

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, malefic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

The term “hydrate” refers to a compound that is complexed to at leastone water molecule. The compounds of the present invention can becomplexed with from 1 to 10 water molecules.

The term “isomers” refers to compounds with the same chemical formulabut which are structurally distinguishable. Certain compounds of thepresent invention possess asymmetric carbon atoms (optical centers) ordouble bonds; the racemates, diastereomers, geometric isomers andindividual isomers are all intended to be encompassed within the scopeof the present invention.

The term “tautomer” refers to one of two or more structural isomerswhich exist in equilibrium and which are readily converted from one formto another. The present invention includes all tautomers andstereoisomers of compounds of the present invention, either in admixtureor in pure or substantially pure form. The compounds of the presentinvention can have asymmetric centers at the carbon atoms, and thereforethe compounds of the present invention can exist in diastereomeric orenantiomeric forms or mixtures thereof. All conformational isomers(e.g., cis and trans isomers) and all optical isomers (e.g., enantiomersand diastereomers), racemic, diastereomeric and other mixtures of suchisomers, as well as solvates, hydrates, isomorphs, polymorphs andtautomers are within the scope of the present invention. Compoundsaccording to the present invention can be prepared using diastereomers,enantiomers or racemic mixtures as starting materials. Furthermore,diastereomer and enantiomer products can be separated by chromatography,fractional crystallization or other methods known to those of skill inthe art.

The terms “pharmaceutically acceptable excipient” and “pharmaceuticallyacceptable carrier” refer to a substance that aids the administration ofan active agent to and absorption by a subject and can be included inthe compositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors and colors, and the like. One of skill in the art will recognizethat other pharmaceutical excipients are useful in the presentinvention.

The term “contacting” refers to the process of bringing into contact atleast two distinct species such that they can react with one another orinteract such that one has an effect on the other. It should beappreciated, however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The terms “treat”, “treating” and “treatment” refer to any indicia ofsuccess in the treatment or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; improving a patient's physical or mental well-being.The treatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation.

The terms “patient” or “subject in need thereof” refers to a livingorganism suffering from or prone to a condition that can be treated byadministration of a pharmaceutical composition as provided herein.Non-limiting examples include humans, other mammals and othernon-mammalian animals.

The terms “disorder” or “condition” refer to a state of being or healthstatus of a patient or subject capable of being treated with thegalectin-1 (gal-1) inhibitors of the present invention. Examples ofdisorders or conditions include, but are not limited to, ovarian cancer,prostate cancer, lung cancer, breast cancer, kidney cancer, pancreaticcancer, colon cancer, and non-small cell lung cancer.

The term “chemotherapeutic agent” refers to a compound or pharmaceuticalcomposition useful for treating or ameliorating cancer. The agent can begiven with a curative intent, with an aim to prolong life, or for thepurpose of reducing symptoms.

III. Methods for Inhibition

In one aspect, the present invention provides several methods forinhibiting respiratory complex III in a cell. The methods includecontacting a cell with a compound that can having the structure:

Each R¹, R², R³, R⁴, R⁵, and R⁶ can independently be hydrogen, C₁₋₆alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and —C(O)—C₁₋₆alkyl, or —C(O)O⁻. The subscripts m and n can each independently beintegers from 0 to 5. The subscript p is an integer from 0 to 4. Thesubscripts q, u, and v can each independently be integers from 0 to 2.The subscript w is an integer from 0 to 3.

In some embodiments, each R¹ of the compound is independently hydrogen,C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and—C(O)—C₁₋₆ alkyl, or —C(O)O⁻. In some embodiments, each R¹ isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl,—NO₂, or —C(O)—C₁₋₆ alkyl. In some embodiments, each R¹ is independentlyC₁₋₆ alkyl, halogen, hydroxyl, or —C(O)—C₁₋₆ alkyl. In some embodiments,each R¹ is independently C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, or hydroxyl. Insome embodiments, each R¹ is independently —NO₂ or C₁₋₆ alkoxy. In someembodiments, each R¹ is independently C₁₋₆ alkoxy or hydroxyl. In someembodiments, each R¹ is independently C₁₋₆ alkoxy. In some embodiments,each R¹ is independently halogen. In some embodiments, R¹ is butyl. Insome embodiments, R¹ of formula is chlorine. In some embodiments, R¹ offormula is hydroxyl. In some embodiments, R¹ is methoxy. In someembodiments, R¹ is —NO₂. In some embodiments, R¹ is methylhydroxy.

In some embodiments, each R² of the compound is independently hydrogen,C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and—C(O)—C₁₋₆ alkyl, or —C(O)O⁻. In some embodiments, each R² isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. In some embodiments, each R²of is independently C₆₋₁₂ aryl, —CN, —NH₂, or —C(O)O—C₁₋₆ alkyl. In someembodiments, each R² is independently —NO₂ or C₁₋₆ alkoxy. In someembodiments, each R² is independently C₁₋₆ alkyl or hydroxyl. In someembodiments, each R² is independently hydroxyl or C(O)O⁻. In someembodiments, each R² is independently C₁₋₆ alkyl. In some embodiments,R² is methyl. In some embodiments, R² is propyl. In some embodiments, R²is hydroxyl. In some embodiments, R² is phenyl. In some embodiments, R²is —CN. In some embodiments, R² is —NH₂. In some embodiments, R² is—C(O)O-ethyl. In some embodiments, R² is —NO₂. In some embodiments, R²is methoxy. In some embodiments, R² is C(O)O⁻.

In some embodiments, each R³ of the compound is independently hydrogen,C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and—C(O)—C₁₋₆ alkyl, or —C(O)O⁻. In some embodiments, each R³ isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆alkoxy, or oxide. In someembodiments, each R³ is independently C₁₋₆ alkyl or oxide. In someembodiments, each R³ is independently C₁₋₆ alkyl. In some embodiments,R³ is C₁₋₆ alkoxy. In some embodiments, each R³ is independentlyhalogen. In some embodiments, R³ is methyl. In some embodiments, R³ ischlorine. In some embodiments, R³ is oxide. In some embodiments, R³ ismethoxy.

In some embodiments, each R⁴ of the compound is independently hydrogen,C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and—C(O)—C₁₋₆ alkyl, or —C(O)O⁻. In some embodiments, each R⁴ isindependently hydrogen or C₁₋₆ alkyl. In some embodiments, each R⁴ isindependently C₁₋₆ alkyl. In some embodiments, each R⁴ is methyl.

In some embodiments, each R⁵ of the compound is independently hydrogen,C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and—C(O)—C₁₋₆ alkyl, or —C(O)O⁻. In some embodiments, each R⁵ isindependently C₁₋₆ alkyl or —C(O)O⁻. In some embodiments, R⁵ is methyl.In some embodiments, R⁵ is —C(O)O⁻. In some embodiments, R⁵ is hydroxyl.

In some embodiments, each R⁶ of the compound is independently hydrogen,C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and—C(O)—C₁₋₆ alkyl, or —C(O)O⁻. In some embodiments, R⁶ is hydroxyl.

In some embodiments, subscripts m and n are each independently integersfrom 0 to 5. In some embodiments, subscript m is 0, 1, 2, 3, 4, or 5. Insome embodiments, subscript m is 3. In some embodiments, subscript m is2. In some embodiments, subscript m is 1. In some embodiments, subscriptm is 0. The subscript n can be 0, 1, 2, 3, 4, or 5. In some embodiments,subscript n is 4. In some embodiments, subscript n is 3. In someembodiments, subscript n is 2. In some embodiments, subscript n is 1. Insome embodiments, subscript n is 0. The subscript p can be an integerfrom 0 to 4. In some embodiments, subscript p is 0, 1, 2, 3, or 4. Insome embodiments, subscript p is 2. In some embodiments, subscript pis 1. In some embodiments, subscript p is 0. In some embodiments, thesubscripts q, u, and v are each independently integers from 0 to 2. Insome embodiments, subscript q is 0, 1, or 2. In some embodiments,subscript q is 2. In some embodiments, subscript q is 0. In someembodiments, subscript u is 0, 1, or 2. In some embodiments, subscript uis 2. In some embodiments, subscript v is 0, 1, or 2. In someembodiments, subscript v is 1. In some embodiments, the subscript w isan integer from 0 to 3. In some embodiments, subscript w is 0, 1, 2, or3. In some embodiments, subscript w is 1.

In some embodiments, the compound has the structure of formula I:

wherein each R¹ can independently be hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula I canindependently be hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl,hydroxyl, —CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formula Ican independently be hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide. EachR⁴ of formula I can independently be hydrogen or C₁₋₆ alkyl. Thesubscripts m and n of formula I can each independently be integers from0 to 5. The subscript p of formula I can be an integer from 0 to 4. Thesubscript q of formula I can be an integer from 0 to 2.

In some embodiments, R¹ of formula I is hydroxyl. In some embodiments,R² of formula I is hydroxyl. In some embodiments, each R³ of formula Iis independently C₁₋₆ alkyl. In some embodiments, R³ of formula I ismethyl. In some embodiments, each R⁴ of formula I is independently C₁₋₆alkyl. In some embodiments, R⁴ of formula I is methyl. In someembodiments, the subscript m of formula I is 2. In some embodiments, thesubscript n of formula I is 2. In some embodiments, the subscript p offormula I is 2. In some embodiments, the subscript q of formula I is 2.In some embodiments, the compound of formula I is:

In some embodiments, the compound has the structure of formula II:

wherein each R¹ can independently be hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula II canindependently be hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl,hydroxyl, —CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. The subscripts m andn of formula II can each independently be integers from 0 to 5.

In some embodiments, each R¹ of formula II is independently C₁₋₆ alkyl,halogen, hydroxyl, or —C(O)—C₁₋₆ alkyl. In some embodiments, R¹ offormula II is butyl. In some embodiments, R¹ of formula II is ethyl. Insome embodiments, R¹ of formula II is —C(O)— methyl. In someembodiments, R¹ of formula II is hydroxyl. In some embodiments, each R²of formula II is independently C₁₋₆ alkyl. In some embodiments, R² offormula II is methyl. In some embodiments, the subscripts m and n offormula II are each independently integers from 2 to 5. In someembodiments, subscript m of formula II is 2. In some embodiments,subscript n of formula II is 5. In some embodiments, subscript n is 4.In some embodiments, the compound of formula II is:

In some embodiments, the compound has the structure of formula III:

wherein each R¹ can independently be hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula III canindependently be hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl,hydroxyl, —CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formulaIII can independently be hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide.The subscripts m and n of formula III can each independently be integersfrom 0 to 5. The subscript p of formula III is an integer from 0 to 4.

In some embodiments, R¹ of formula III is halogen. In some embodiments,R¹ of formula III is chlorine. In some embodiments, each R³ of formulaIII is independently C₁₋₆ alkyl or oxide. In some embodiments, R³ offormula III is methyl. In some embodiments, R³ of formula III is oxide.In some embodiments, subscript m of formula III is 1. In someembodiments, subscript n of formula III is 0. In some embodiments,subscript p of formula III is 2. In some embodiments, the compound offormula III is:

In some embodiments, the compound has the structure of formula IV:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula IV isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. The subscripts m and n offormula IV are each independently integers from 0 to 5.

In some embodiments, R¹ of formula IV is halogen. In some embodiments,R¹ of formula IV is chlorine. In some embodiments, each R² of formula IVis independently C₆₋₁₂ aryl, —CN, —NH₂, or —C(O)O—C₁₋₆ alkyl. In someembodiments, R² of formula IV is phenyl. In some embodiments, R² offormula IV is —CN. In some embodiments, R² of formula IV is —NH₂. Insome embodiments, R² of formula IV is —C(O)O-ethyl. In some embodiments,subscript m of formula IV is 1. In some embodiments, subscript n offormula IV is 4. In some embodiments, the compound of formula IV is:

In some embodiments, the compound has the structure of formula V:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula V isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formula V isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide. Each R⁴ offormula V is independently hydrogen or C₁₋₆ alkyl. Subscripts m and n offormula V are each independently integers from 0 to 5. Subscript p offormula V is an integer from 0 to 4. Subscript q of formula V is aninteger from 0 to 2.

In some embodiments, each R¹ of formula V is independently C₁₋₆ alkoxy.In some embodiments, R¹ of formula V is methoxy. In some embodiments,each R² of formula V is independently C₁₋₆ alkyl or hydroxyl. In someembodiments, R² of formula V is methyl. In some embodiments, R² offormula V is hydroxyl. In some embodiments, subscript m of formula V is3. In some embodiments, subscript n of formula V is 3. In someembodiments, subscript p of formula V is 0. In some embodiments,subscript q of formula V is 0. In some embodiments, the compound offormula V is:

In some embodiments, the compound has the structure of formula VI:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula VI isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. The subscripts m and n offormula VI are each independently integers from 0 to 5.

In some embodiments, each R¹ of formula VI is independently —NO₂ or C₁₋₆alkoxy. In some embodiments, R¹ of formula VI is —NO₂. In someembodiments, R¹ of formula VI is methoxy. In some embodiments, each R²of formula VI is independently —NO₂ or C₁₋₆ alkoxy. In some embodiments,R² of formula VI is —NO₂. In some embodiments, R² of formula VI ismethoxy. In some embodiments, subscript m of formula VI is 1. In someembodiments, subscript n of formula VI is 1. In some embodiments, thecompound of formula VI is:

In some embodiments, the compound has the structure of formula VII:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula VII isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formula VII isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide. Thesubscripts m and n of formula VII are each independently integers from 0to 5.

In some embodiments, R² of formula VII is C(O)O⁻. In some embodiments,subscript m is 0. In some embodiments, subscript n of formula VII is 1.In some embodiments, subscript p of formula VII is 0. In someembodiments, the compound of formula VII is:

In some embodiments, the compound has the structure of formula VIII:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula VIII isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formula VIII isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide. Each R⁴ offormula VIII is independently hydrogen or C₁₋₆ alkyl. The subscripts mand n of formula VIII are each independently integers from 0 to 5. Thesubscript p of formula VIII is an integer from 0 to 4. The subscript qof formula VIII is an integer from 0 to 2.

In some embodiments, each R¹ of formula VIII is independently C₁₋₆alkoxy or hydroxyl. In some embodiments, R¹ of formula VIII is methoxy.In some embodiments, R¹ of formula VIII is hydroxyl. In someembodiments, each R² of formula VIII is independently C₁₋₆ alkoxy orhydroxyl. In some embodiments, R² of formula VIII is methoxy. In someembodiments, R² of formula VIII is hydroxyl. In some embodiments, R³ offormula VIII is C₁₋₆ alkoxy. In some embodiments, R³ of formula VIII ismethoxy. In some embodiments, subscript m of formula VIII is 2. In someembodiments, subscript n of formula VIII is 2. In some embodiments,subscript p of formula VIII is 1. In some embodiments, subscript q offormula VIII is 0. In some embodiments, the compound of formula VIII is:

In some embodiments, the compound has the structure of formula IX:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula IX isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formula IX isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide. Each R⁴ offormula IX is independently hydrogen or C₁₋₆ alkyl. The subscripts m andn of formula IX are each independently integers from 0 to 5. Thesubscript p of formula IX is an integer from 0 to 4. The subscript q offormula IX is an integer from 0 to 2.

In some embodiments, subscript m of formula IX is 0. In someembodiments, subscript n of formula IX is 0. In some embodiments,subscript p of formula IX is 0. In some embodiments, subscript q orformula IX is 0. In some embodiments, the compound of formula IX is:

In some embodiments the compound has the structure of formula X:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula X isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formula X isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide. Thesubscripts m and n of formula X are each independently integers from 0to 5. The subscript p of formula X is an integer from 0 to 4.

In some embodiments, each R¹ of formula X is independently C₁₋₆ alkyl,C₁₋₆ alkylhydroxy, or hydroxyl. In some embodiments, R¹ of formula X ismethyl. In some embodiments, R¹ of formula X is hydroxymethyl. In someembodiments, R¹ of formula X is hydroxyl. In some embodiments, each R²of formula X is independently C₁₋₆ alkyl. In some embodiments, R² offormula X is propyl. In some embodiments, subscript m of formula X is 3.In some embodiments, subscript n of formula X is 2. In some embodiments,the compound of formula X is:

In some embodiments, the compound has the structure of formula XI:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula XI isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. The subscripts m and n offormula XI are each independently integers from 0 to 5. The subscript wof formula XI is an integer from 0 to 3.

In some embodiments, subscript m of formula XI is 0. In someembodiments, subscript n of formula XI is 0. In some embodiments,subscript w of formula XI is 1. In some embodiments, the compound offormula XI is:

In some embodiments, the compound has the structure of formula XII:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula XII isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formula XII isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide. Each R⁴ offormula XII is independently hydrogen or C₁₋₆ alkyl. Each R⁵ of formulaXII is independently hydrogen, C₁₋₆ alkyl, hydroxyl, or C(O)O⁻. Thesubscripts m and n of formula XII are each independently integers from 0to 5. The subscript p of formula XII is an integer from 0 to 4. Thesubscripts q and u of formula XII are each independently integers from 0to 2.

In some embodiments, each R³ of formula XII is independently C₁₋₆ alkyl.In some embodiments, R³ of formula XII is methyl. In some embodiments,each R⁴ of formula XII is independently C₁₋₆ alkyl. In some embodiments,R⁴ of formula XII is methyl. In some embodiments, each R⁵ of formula XIIis independently C₁₋₆ alkyl or —C(O)O⁻. In some embodiments, R⁵ offormula XII is methyl. In some embodiments, R⁵ of formula XII is—C(O)O⁻. In some embodiments, subscript m of formula XII is 0. In someembodiments, subscript n of formula n is 0. In some embodiments,subscript p of formula XII is 2. In some embodiments, subscript q offormula XII is 2. In some embodiments, subscript u of formula XII is 2.In some embodiments, the compound of formula XII is:

In some embodiments, the compound has the structure of formula XIII:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula XIII isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. Each R³ of formula XIII isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, or oxide. Each R⁴ offormula XIII is independently hydrogen or C₁₋₆ alkyl. Each R⁵ of formulaXIII is independently hydrogen, C₁₋₆ alkyl, hydroxyl, or C(O)O⁻. Each R⁶of formula XIII is hydrogen or hydroxyl. The subscripts m and n offormula XIII are each independently integers from 0 to 5. The subscriptp of formula XIII is an integer from 0 to 4. The subscripts q, u, and vof formula XIII are each independently integers from 0 to 2.

In some embodiments, R¹ of formula XIII is hydroxyl. In someembodiments, R² of formula XIII is hydroxyl. In some embodiments, R⁵ offormula XIII is hydroxyl. In some embodiments, R⁶ of formula XIII ishydroxyl. In some embodiments, each R³ of formula XIII is independentlyC₁₋₆ alkyl. In some embodiments, R³ of formula XIII is methyl. In someembodiments, each R⁴ of formula XIII is independently C₁₋₆ alkyl. Insome embodiments, R⁴ of formula XIII is methyl. In some embodiments,subscript m of formula XIII is 2. In some embodiments, subscript n offormula XIII is 2. In some embodiments, subscript p of formula XIII is2. In some embodiments, subscript q of formula XIII is 2. In someembodiments, subscript u of formula XIII is 1. In some embodiments,subscript v of formula XIII is 1. In some embodiments, the compound offormula XIII is:

In some embodiments, the compound has the structure of formula XIV:

wherein each R¹ is independently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,halogen, hydroxyl, —NO₂, or —C(O)—C₁₋₆ alkyl. Each R² of formula XIV isindependently hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, or —C(O)O⁻. The subscripts m and n offormula XIV are each independently integers from 0 to 5.

In some embodiments, each R¹ of formula XIV is C₁₋₆ alkyl. In someembodiments, R¹ of formula XIV is butyl. In some embodiments, each R² offormula XIV is independently hydroxyl or C(O)O⁻. In some embodiments, R²of formula XIV is hydroxyl. In some embodiments, R² of formula XIV isC(O)O⁻. In some embodiments, subscript m of formula XIV is 1. In someembodiments, subscript n of formula XIV is 2. In some embodiments, thecompound of formula XIV is:

In some embodiments, the compound has the structure of any of theformulas of FIG. 2. Each R of FIG. 2 can independently be be hydrogen,C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁-6 haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, or—C(O)—C₁₋₆ alkyl, or —C(O)O⁻. Each X of FIG. 2 can independently be S,N, or O.

The compounds of the present invention can also be the salts and isomersthereof. In some embodiments, the compounds of the present inventioninclude the salt forms thereof. Examples of applicable salt formsinclude hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures), succinates, benzoates and salts with amino acids such asglutamic acid. These salts may be prepared by methods known to thoseskilled in art. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of acceptable acid addition salts include those derived frominorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromorganic acids like acetic, propionic, isobutyric, maleic, malonic,benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic,benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, andthe like. Also included are salts of amino acids such as arginate andthe like, and salts of organic acids like glucuronic or galactunoricacids and the like (see, for example, Berge et al. (1977) Journal ofPharmaceutical Science 66:1). Certain specific compounds of the presentinvention contain basic acidic functionalities that allow the compoundsto be converted into base addition salts. Additional information onsuitable pharmaceutically acceptable salts can be found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, which is incorporated herein by reference.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisometric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)-for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques.

Isomers include compounds having the same number and kind of atoms, andhence the same molecular weight, but differing in respect to thestructural arrangement or configuration of the atoms.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention. Tautomerrefers to one of two or more structural isomers which exist inequilibrium and which are readily converted from one isomeric form toanother.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, the compounds of the present invention may alsocontain unnatural proportions of atomic isotopes at one or more of theatoms that constitute such compounds. For example, the compounds of thepresent invention may be radiolabeled with radioactive isotopes, such asfor example deuterium (²H), tritium (³H), iodine-125 (¹²⁵I), carbon-13(¹³C), or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

IV. Methods for Treatment

In one aspect, the present invention provides several methods fortreatment of cancer in a subject. The methods include administering to asubject in need of such treatment, a therapeutically effective amount ofa compound having the structure:

Each R¹, R², R³, R⁴, R⁵, and R⁶ can independently be hydrogen, C₁₋₆alkyl, C₁₋₆ alkylhydroxy, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,C₆₋₁₂ aryl, halogen, hydroxyl, oxide, —CN, —NH₂, —NO₂, and —C(O)—C₁₋₆alkyl, or —C(O)O⁻. The subscripts m and n can each independently beintegers from 0 to 5. The subscript p is an integer from 0 to 4. Thesubscripts q, u, and v can each independently be integers from 0 to 2.The subscript w is an integer from 0 to 3.

In some embodiments, the compound administered for treatment has any oneof the structures of formulas I-XIV described above. In someembodiments, more than one compound is administered for treatment. Insome embodiments, the more than one administered compounds include twoor more compounds each having a structure of one of formulas I-XIVdescribed above.

In some embodiments, the subject is a patient suffering from cancer. Insome embodiments, the patient is a human. In some embodiments, thepatient suffers from more than one cancer. Examples of cancers suitablefor treatment with the present invention include, but are not limitedto, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, anal cancer, appendix cancer, astrocytoma, basal-cellcarcinoma, bile duct cancer, bladder cancer, bone tumor, brainstemglioma, brain cancer, cerebellar astrocytoma, cerebral astrocytoma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas, Burkitt's lymphoma, central nervous system lymphoma,cerebellar astrocytoma, cervical cancer, chondrosarcoma, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma,desmoplastic small round cell tumor, endometrial cancer, ependymoma,epitheliod hemangioendothelioma (EHE), esophageal cancer, Ewing'ssarcoma, extracranial germ cell tumor, extragonadal germ cell tumor,extrahepatic bile duct cancer, eye cancer, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinalcarcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor,gestational trophoblastic tumor, gastric carcinoid, hairy cell leukemia,head and neck cancer, heart cancer, hepatocellular cancer, Hodgkinlymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma,childhood, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma,kidney cancer, laryngeal cancer, leukaemias, lip and oral cavity cancer,liposarcoma, liver cancer, non-small cell lung cancer, small-cell lungcancer, lymphomas, macroglobulinemia, male breast cancer, malignantfibrous histiocytoma of bone, medulloblastoma, melanoma, Merkel cellcancer, mesothelioma, metastatic squamous neck cancer, mouth cancer,multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, myelodysplastic syndromes, myelogenous leukemia, myeloidleukemia, adult acute, myeloproliferative disorders, chronic, myxoma,nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma,neuroblastoma, non-Hodgkin lymphoma, oligodendroglioma, oral cancer,oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelialcancer, ovarian germ cell tumor, ovarian low malignant potential tumor,pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroidcancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealastrocytoma, pineal germinoma, pineoblastoma, supratentorial primitiveneuroectodermal tumors, pituitary adenoma. plasma cell neoplasia,pleuropulmonary blastoma, primary central nervous system lymphoma,prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, Ewing sarcoma, Kaposi sarcoma,soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skincancer, melanoma Merkel cell skin carcinoma, small intestine cancer,squamous cell carcinoma, squamous neck cancer, stomach cancer, cutaneousT-Cell lymphoma, testicular cancer, throat cancer, thymoma, thyroidcancer, transitional cell cancer of the renal pelvis and ureter,trophoblastic tumor, gestational, urethral cancer, uterine cancer,vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilmstumor.

The compounds and compositions of the present invention can be deliveredby any suitable means, including oral, parenteral and topical methods.Transdermal administration methods, by a topical route, can beformulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the compounds and compositions of the presentinvention. The unit dosage form can be a packaged preparation, thepackage containing discrete quantities of preparation, such as packetedtablets, capsules, and powders in vials or ampoules. Also, the unitdosage form can be a capsule, tablet, cachet, or lozenge itself, or itcan be the appropriate number of any of these in packaged form.

The compounds and compositions of the present invention can beco-administered with other agents. Co-administration includesadministering the compound or composition of the present inventionwithin 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of the otheragent. Co-administration also includes administering simultaneously,approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30minutes of each other), or sequentially in any order. Moreover, thecompounds and compositions of the present invention can each beadministered once a day, or two, three, or more times per day so as toprovide the preferred dosage level per day.

In some embodiments, co-administration can be accomplished byco-formulation, i.e., preparing a single pharmaceutical compositionincluding the compounds and compositions of the present invention andany other agent. Alternatively, the various components can be formulatedseparately.

The compounds and compositions of the present invention, and any otheragents, can be present in any suitable amount, and can depend on variousfactors including, but not limited to, weight and age of the subject,state of the disease, etc. Suitable dosage ranges include from about 0.1mg to about 10,000 mg, or about 1 mg to about 1000 mg, or about 10 mg toabout 750 mg, or about 25 mg to about 500 mg, or about 50 mg to about250 mg. Suitable dosages also include about 1 mg, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg.

The compounds of the present invention can be administered at anysuitable frequency, interval and duration. For example, the compound ofthe present invention can be administered once an hour, or two, three ormore times an hour, once a day, or two, three, or more times per day, oronce every 2, 3, 4, 5, 6, or 7 days, so as to provide the preferreddosage level. When the compound of the present invention is administeredmore than once a day, representative intervals include 5, 10, 15, 20,30, 45 and 60 minutes, as well as 1, 2, 4, 6, 8, 10, 12, 16, 20, and 24hours. The compound of the present invention can be administered once,twice, or three or more times, for an hour, for 1 to 6 hours, for 1 to12 hours, for 1 to 24 hours, for 6 to 12 hours, for 12 to 24 hours, fora single day, for 1 to 7 days, for a single week, for 1 to 4 weeks, fora month, for 1 to 12 months, for a year or more, or even indefinitely.

V. Pharmaceutical Compositions

In some embodiments, the present invention provides a pharmaceuticalcomposition including a pharmaceutically acceptable excipient and acompound of the present invention. In some embodiments, the compositionalso includes an additional chemotherapeutic agent.

Chemotherapeutic Agents

Chemotherapeutic agents suitable for use with the present inventioninclude those agents that are useful for treating or ameliorating cancerand include, but are not limited to, aldesleukin, alectinib anaplasticlymphoma kinase, cabozantinib, elotuzumab, fluoxymesterone, iobenguane,imiquimod, interferon, ixazomib, lanreotide, lentinan, mitotane,nab-paclitaxel, necitumumab, octreotide, somatostatin, omacetaxine,sipuleucel-T, tegafur/gimeracil/oteracil and tegafur/uracil.

Additional chemotherapeutic agents suitable for use with the presentinvention include, but are not limited to, azacitidine, capecitabine,carmofur, cladribine, clofarabine, cytarabine, decitabine, floxuridine,fludarabine, fluorouracil, gemcitabine, mercaptopurine, nelarabine,pentostatin, tegafur, tioguanine, trifluridine/tipiracil, methotrexate,pemetrexed, pralatrexate, raltitrexed, hydroxycarbamide, irinotecan,topotecan, daunorubicin, doxorubicin, epirubicin, idarubicin,mitoxantrone, valrubicin, etoposide, teniposide, cabazitaxel, docetaxel,paclitaxel, vinblastine, vincristine, vindesine, vinflunine,vinorelbine, bendamustine, busulfan, carmustine, chlorambucil,chlormethine, cyclophosphamide, dacarbazine, fotemustine, ifosfamide,lomustine, melphalan, streptozotocin, temozolomide, trabectedin,carboplatin, cisplatin, nedaplatin, oxaliplatin, altretamine, bleomycin,bortezomib, carfilzomib, dactinomycin, eribulin, estramustine,ixabepilone, mitomycin, procarbazine, abarelix, abiraterone,anastrozole, bicalutamide, cyproterone, degarelix, enzalutamide,exemestane, flutamide, fulvestrant, goserelin, histrelin, letrozole,leuprolide, mifepristone, nilutamide, tamoxifen, toremifene,triptorelin, ibritumomab tiuxetan, radium Ra 223 dichloride,strontium-89, samarium (153 Sm) lexidronam, tositumomab, ado-trastuzumabemtansine, alemtuzumab, bevacizumab, blinatumomab, brentuximab vedotin,cetuximab, daratumumab, denosumab, dinutuximab, gemtuzumab ozogamicin,ibritumomab tiuxetan, ipilimumab, nivolumab, obinutuzumab, ofatumumab,panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab,tositumomab, trastuzumab, afatinib, aflibercept, axitinib, bosutinib,cobimetinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinibl,lapatinibl, lenvatinibl, nilotinib, osimertinib, pazopanib, ponatinib,regorafenib, ruxolitinib, sorafenib, sunitinib, trametinib, vandetanib,everolimus, temsirolimus, alitretinoin, bexarotene, isotretinoin,tamibarotene, tretinoin, lenalidomide, pomalidomide, thalidomide,belinostat, panobinostat, romidepsin, valproate, vorinostat, anagrelide,arsenic trioxide, asparaginase, Bacillus Calmete-Guerin vaccine,ceritinib, dabrafenib, denileukin diftitox, idelalisib, ibrutinib,olaparib, palbociclib, sonidegib, talimogene laherparepvec, vemurafenib,and vismodegib.

The chemotherapeutic agents of the present invention also include thesalts, hydrates, solvates and prodrug forms. The compounds of thepresent invention also include the isomers and metabolites of thosedescribed above.

Salts include, but are not limited, to sulfate, citrate, acetate,oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, phosphonic acid, isonicotinate, lactate, salicylate, citrate,tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Other saltsinclude, but are not limited to, salts with inorganic bases includingalkali metal salts such as sodium salts, and potassium salts; alkalineearth metal salts such as calcium salts, and magnesium salts; aluminumsalts; and ammonium salts. Other salts with organic bases include saltswith diethylamine, diethanolamine, meglumine, andN,N′-dibenzylethylenediamine.

The neutral forms of the chemotherapeutic agents can be regenerated bycontacting the salt with a base or acid and isolating the parentanti-inflammatory glucocorticosteroid in the conventional manner. Theparent form of the anti-inflammatory glucocorticosteroid differs fromthe various salt forms in certain physical properties, such assolubility in polar solvents, but otherwise the salts are equivalent tothe parent form of the compound for the purposes of the presentinvention.

Certain chemotherapeutic agents of the present invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain chemotherapeutic agents of the present invention possessasymmetric carbon atoms (optical centers) or double bonds; theenantiomers, racemates, diastereomers, tautomers, geometric isomers,stereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)- or, as (D)- or (L)-for amino acids, andindividual isomers are encompassed within the scope of the presentinvention. The compounds of the present invention do not include thosewhich are known in art to be too unstable to synthesize and/or isolate.The present invention is meant to include compounds in racemic andoptically pure forms. Optically active (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques.

The present invention also provides chemotherapeutic agents which are ina prodrug form. Prodrugs of the compounds described herein are thosecompounds that readily undergo chemical changes under physiologicalconditions to provide the compounds of the present invention.Additionally, prodrugs can be converted to the compounds of the presentinvention by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to the compounds of thepresent invention when placed in a transdermal patch reservoir with asuitable enzyme or chemical reagent.

VI. Formulations

The compositions of the present invention can be prepared in a widevariety of oral, parenteral and topical dosage forms. Oral preparationsinclude tablets, pills, powder, dragees, capsules, liquids, lozenges,cachets, gels, syrups, slurries, suspensions, etc., suitable foringestion by the patient. The compositions of the present invention canalso be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compositions described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompositions of the present invention can be administered transdermally.The compositions of this invention can also be administered byintraocular, intravaginal, and intrarectal routes includingsuppositories, insufflation, powders and aerosol formulations (forexamples of steroid inhalants, see Rohatagi (1995) J Clin. Pharmacol.35:1187; and Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107).Accordingly, the present invention also provides pharmaceuticalcompositions including a pharmaceutically acceptable carrier orexcipient and a compound of the present invention.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances, which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.(“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the compounds of the present invention.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; talc; pectin; dextrin; starch;tragacanth; a low melting wax; cocoa butter; carbohydrates; sugarsincluding, but not limited to, lactose, sucrose, mannitol, or sorbitol,starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins including, but not limited to, gelatin and collagen. Ifdesired, disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can contain thecompounds of the present invention mixed with a filler or binders suchas lactose or starches, lubricants such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the compounds of thepresent invention may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycol withor without stabilizers.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the compoundsof the present invention are dispersed homogeneously therein, as bystirring. The molten homogeneous mixture is then poured into convenientsized molds, allowed to cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe compounds of the present invention in water and adding suitablecolorants, flavors, stabilizers, and thickening agents as desired.Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partialester derived from a fatty acid and a hexitol (e.g., polyoxyethylenesorbitol mono-oleate), or a condensation product of ethylene oxide witha partial ester derived from fatty acid and a hexitol anhydride (e.g.,polyoxyethylene sorbitan mono-oleate). The aqueous suspension can alsocontain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose, aspartame orsaccharin. Formulations can be adjusted for osmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can be formulated by suspending the compounds of thepresent invention in a vegetable oil, such as arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin;or a mixture of these. The oil suspensions can contain a thickeningagent, such as beeswax, hard paraffin or cetyl alcohol. Sweeteningagents can be added to provide a palatable oral preparation, such asglycerol, sorbitol or sucrose. These formulations can be preserved bythe addition of an antioxidant such as ascorbic acid. As an example ofan injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther.281:93. The pharmaceutical formulations of the invention can also be inthe form of oil-in-water emulsions. The oily phase can be a vegetableoil or a mineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. The emulsion can also contain sweetening agentsand flavoring agents, as in the formulation of syrups and elixirs. Suchformulations can also contain a demulcent, a preservative, or a coloringagent.

The compositions of the present invention can also be delivered asmicrospheres for slow release in the body. For example, microspheres canbe formulated for administration via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao (1995) J. Biomater Sci. Polym. Ed. 7:623); as biodegradable andinjectable gel formulations (see, e.g., Gao (1995) Pharm. Res. 12:857);or, as microspheres for oral administration (see, e.g., Eyles (1997) J.Pharm. Pharmacol. 49:669). Both transdermal and intradermal routesafford constant delivery for weeks or months.

In another embodiment, the compositions of the present invention can beformulated for parenteral administration, such as intravenous (IV)administration or administration into a body cavity or lumen of anorgan. The formulations for administration will commonly comprise asolution of the compositions of the present invention dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well known sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of the compositions ofthe present invention in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight, andthe like, in accordance with the particular mode of administrationselected and the patient's needs. For IV administration, the formulationcan be a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In another embodiment, the formulations of the compositions of thepresent invention can be delivered by the use of liposomes which fusewith the cellular membrane or are endocytosed, i.e., by employingligands attached to the liposome, or attached directly to theoligonucleotide, that bind to surface membrane protein receptors of thecell resulting in endocytosis. By using liposomes, particularly wherethe liposome surface carries ligands specific for target cells, or areotherwise preferentially directed to a specific organ, one can focus thedelivery of the compositions of the present invention into the targetcells in vivo. (See, e.g., Al-Muhammed (1996) J. Microencapsul. 13:293;Chonn (1995) Curr. Opin. Biotechnol. 6:698; and Ostro (1989) Am. I Hosp.Pharm. 46:1576).

Lipid-based drug delivery systems include lipid solutions, lipidemulsions, lipid dispersions, self-emulsifying drug delivery systems(SEDDS) and self-microemulsifying drug delivery systems (SMEDDS). Inparticular, SEDDS and SMEDDS are isotropic mixtures of lipids,surfactants and co-surfactants that can disperse spontaneously inaqueous media and form fine emulsions (SEDDS) or microemulsions(SMEDDS). Lipids useful in the formulations of the present inventioninclude any natural or synthetic lipids including, but not limited to,sesame seed oil, olive oil, castor oil, peanut oil, fatty acid esters,glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®,Capryol®, Capmul®, Captex®, and Peceol®.

In another embodiment, the formulations of the compositions of thepresent invention can be delivered by the use of nanoparticles. Forexample, the use of spin-labeled fluorophores is discussed in Li et al.(2012) ACS Nano. 6:9485. Nanoparticles have emerged as a major class ofvehicles to deliver conventional anticancer drugs. Nanoparticle drugdelivery systems offer several distinct advantages, such as controlledrelease and prolonged circulation time, as well as passive and activetumor targeting (Cabral et al. (2011) Nat. Nanotechnol. 6:815; Gref etal. (1994) Science 263:1600; Liu and Allen (2006) Curr. Pharm. 12:4685;and Li et al. (2009) Nanotechnology 20:065104). In some embodiments, thecompound is hydrophobic and can be easily loaded inside a nanomicelle.The nanomicelle can comprise non-crosslinked micellar nanoparticles(NCMN). The nanomicelle can comprise disulfide-crosslinked micellarnanoparticles (DCMN).

In some embodiments, the formulations of the compositions of the presentinvention comprise solubility aids. The solubility aid can be, forexample, a cyclodextrin. The use of cyclodextrins as solubility aids forhighly water-insoluble steroids is discussed in U.S. Patent ApplicationPublication Nos. US 2015/0018327 and US 2015/0313915. The cyclodextrincan be, for example, a β-cyclodextrin. In some embodiments, thecyclodextrin is a sulfo butyl ether β-cyclodextrin.

VII. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1. Preliminary in Silico Screen of Ligand Binding to the NQ-Siteof Respiratory Complex III

A series of virtual screening computations was conducted with 1957ligands retrieved from the ZINC database which is freely available fromthe Shoichet Laboratory at the University of California, San Francisco(http://zinc.docking.org/; Irwin and Shiochet (2005) J. Chem. Inf. Model45:177). The subset was processed further by AutoDockTools utility(prepareligand4.py; Forli et al. (2016) Nature Protocols 11:905) to addany missing polar hydrogens while preserving the original partialcharges and to convert from Syby1 mol2 format to Autodock PDBQT format.

Virtual screening calculations were carried out using PyRx0.9.2(Dallakyan et al. (2015) Methods Mol. Biol. 1263:243) utilizing bothLamarckian Genetic algorithm (LG) search method in Autodock 4.2 andAutodock Vina 4.2 advanced gradient optimization search method which wasproved to be more accurate than Autodock (Trott and Olson (2010) 1Comput. Chem. 31:455). Default settings were used for Autodock Vina. ForAutodock LG search method, we used the default settings except for thefollowing docking parameters of 100 for ga_run, 10,000,000 forga_num_evals and 1.5 for tstep.

Each of the candidate ligands from the docking calculations was into thenovel NQ-site of the previously energy-minimized bc₁ structure (PDB ID:1BE3). Ligand insertion was accomplished by structural superposition ofthe docked cytochrome b chain (with the ligand) and the energy-minimizedbc₁ structure using the Chimera program (Pettersen et al. (2004) J.Comput. Chem. 25:1605) and the default settings for Needleman-Wunschalgorithm with BLOSUM-62. Subsequently, the whole system was energyminimized using CHARMM PARAM36 (Klauda et al. (2010) J. Phys. Chem. B114:7830) force field for 300,000 steps with periodic boundary conditionand then equilibrated for 100 ps. Molecular dynamics simulation was runon the whole system for 50 nanoseconds. Parameterization of thedifferent discovered ligands were calculated using SwissParam tool(www.swissparam.ch; Zoete et al. (2011) J. Comput. Chem. 32:2359).Intermolecular interaction energies of the different ligands werecomputed based on MM-PBSA free-energy calculation method using theGROMACS g_mmpbsa tool (Kumari et al. (2014) 54:1951) where differentenergy terms (electrostatic energy Eerec, van-der Waals energy Evdw andnon-electrostatic solvation energy using the solvent-accessible surfacearea EsASA) were computed based on the force field parameters. Polarsolvation energy (Eporar1 was calculated using the AdaptivePossion-Boltzmann Solver (APB S) tool (www.poissonboltzmann.org; Bakeret al. (2001) Proc. Natl. Acad. Sci. 107:19157).

To account for flexibility of the NQ-binding site and the differentpossible conformations of the Phe90 residue, we performed the dockingcalculation using both fixed and flexible residues (Phe90, Phe91, Ile92,Leu94 and Tyr95) as shown in FIG. 3. From the data shown in FIG. 3, only39 ligands show a preferential binding by at least 1 kcal/mol at NQ-sitein the flexible-residues docking mode compared to the fixed mode in bothdocking search algorithms. In this way it was ensured that those pickedligands adjust the NQ-site residues and hence bind more firmly. Suchmodification of the NQ-site residues is desirable because it affects thePhe90 internal switch orientation, which is our ultimate target. Theinfluence of those picked ligands on the orientation of Phe90 wassubsequently investigated, and those ligands were chosen which orientPhe90 residue away from the intra-heme b region. In addition, dockingcalculations were performed of the QH₂ at NQ-site as above (indicated bywhite line in FIG. 3) to exclude the ligands that have flexible-modebinding energy higher than that of the QH₂ molecule at the NQ-site(which is on average around −8.75 kcal/mol) in either Vina or LG searchmethods. The result was 30 ligands which have both preferential bindingat the NQ-site compared to QH₂ molecule and induce the desiredconformational change of Phe90 residue.

The ligand search was further restricted such that the candidate ligandswould not compete with QH₂ molecule for binding at Q_(o) site. This isto minimize any possible inhibition of the enzyme by those ligands so asto retain heme b_(L) reduction by QH₂, while at the same time shuttingdown heme b_(L) to heme b_(H) electron transfer and hence allowing forROS production. Further docking analysis of the resultant 30 ligandsinto the active Q_(o) site (as it exists in PDB 1NTZ) reduced the numberof ligands down to 6 candidates. As shown in FIG. 4 and listed in Table1, those 6 candidate ligands exhibit binding energy lower than 8.8kcal/mol and hence would bind preferentially at NQ-site, but not atQ_(o) site. The chemical structures of those ligands are shown in FIG.5.

TABLE 1 Vina and Lamarkian Genetic docking results for the 6 candidateligands and QH2 molecule at NQ and Qo sites. Binding Energy at NQ Site(kcal/mol) Binding Energy at Vina Method LG Method Q_(o) Site (kcal/mol)Ligand Fix Flex Fix Flex Vina Method LG Method ZINC01661542 −8.6 −10.4−7.2 −8.7 −8.0 −7.2 ZINC01691943 −7.6 −8.7 −8.3 −9.4 −8.8 −8.3ZINC01748097 −8.7 −10.1 −8.3 −9.8 −8.3 −8.3 ZINC17465983 −8.3 −9.5 −7.5−12.4 −7.8 −7.5 ZINC17147424 −7.5 −8.6 −8.6 −11.3 −8.1 −8.6 ZINC17147426−7.8 −8.8 −8.2 −10.9 −8.4 −8.2 QH₂ −7.9 −8.5 −8.2 −9.0 −8.5 −8.3

Tunneling calculations were performed as described in Hayashi andStuchebrukhov (2010) Proc. Natl. Acad. Sci. 107:19157; and Hagras andStuchebrukhov (2015) J. Phys. Chem. B. 119:7712. The initial guessmolecular orbitals (MOs) were generated using unrestrictedBroken-Symmetry (BS) B3LYP method of Gaussian 09 package (Frish et al.(2009) Gaussian 09), employing ZINDO basis set for valence electrons andpseudo-potentials for core electrons. The pruned systems werepartitioned into donor, bridge, and acceptor(s) fragments of differentcharges and spin multiplicities, and consequently the tunneling electronwas localized either on the donor or the acceptor site. The computedinitial-guess MOs were then utilized in a subsequent BS-ZINDOcalculation to obtain the corresponding BS donor and acceptor diabaticground states,

|Ψ_(D)

=|φ_(1,σ) ^(D), . . . ,φ_(N,σ) ^(D)

,|Ψ_(A)

=|φ_(1,σ) ^(A), . . . ,φ_(N,σ) ^(A)

.

The Hartree-Fock approximation assumed above, was validated in ourrecent study (Hagras and Stuchebrukhov (2015)). To simplify thetunneling calculation, the reduction from a fully multi-electronicpicture to a one-electron approximation was accomplished by usingbi-orthogonalization scheme of corresponding orbitals (Stuchebrukhov(2003) J. Chem. Phys. 118:7898; and Stuchebrukhov (2003) Theor. Chem.Acc. 110:291). The donor and acceptor orbitals with smallest overlap,and hence with most significant change during the tunneling transition,represent the tunneling pair of the one-electron approximation; theremaining corresponding (core) orbitals experience only relatively smallchange in the tunneling transition, with almost unit overlap, andcontribute to the tunneling matrix element via the electronicFranck-Condon factor (Hagras and Stuchebrukhov (2015)). The transformedcorresponding orbitals have the following form,

|Ψ′_(D)

=|ξ_(1,α) ^(D), . . . ,ξ_(l,α) ^(D),ξ_(1,β) ^(D), . . . ,ξ_(t,β) ^(D)

,

|Ψ′_(A)

=|ξ_(1,α) ^(A), . . . ,ξ_(l,α) ^(A),ξ_(1,β) ^(A), . . . ,ξ_(t,β) ^(A)

Ψ′_(D)|Ψ′_(A)

=

ξ_(i,σ) ^(D)|ξ_(j,σ) ^(A)

=δ_(ij) s _(i) ^(σ)

The product of the overlaps s_(i) ^(σ) of the core orbitals forms theelectronic Franck-Condon factor

${\prod\limits_{j}^{l}{s_{i}^{\alpha}{\prod\limits_{j \neq t}^{t}s_{j}^{\beta}}}},$

where we assume that the tunneling electron has a β-spin, and thetunneling orbital index “t” is the last one of the β-spin orbitals.

Since the ZINDO canonical MOs are represented in a basis set which isorthogonalized (using Löwdin-orthogonalization (Lowdin (1947) Ark. Mat.Astr. Fys. A 35:9) or other schemes) and therefore delocalized over manyatoms, the localized atomic picture of inter-atomic tunneling currentsin a tunneling transition is lost in this basis set. To conduct thetunneling current calculations, the atomic basis set localization needstherefore to be restored. By introducing the localized atomic basis setand the Mulliken type coarse-graining of the tunneling current, thetunneling transition flux is expressed in terms of the interatomiccurrents, which (approximately) has the following form (Stuchebrukhov(1998) J. Chem. Phys. 108:8510):

$J_{ab} = {\prod\limits_{i}{s_{i}^{\alpha}{\prod\limits_{j \neq t}{s_{j}^{\beta}{\sum\limits_{\nu \in a}{\sum\limits_{\mu \in b}{\left( {H_{\nu\mu} - {E_{0}S_{\nu\mu}}} \right)\left( {\theta_{\mu\nu} - \theta_{\nu\mu}} \right)}}}}}}}$

Here v and μ are the atomic orbitals of atoms a and b; θ_(vμ)=A_(v)D_(μ)where D_(μ) and A_(v) are the expansion coefficients of the donor andacceptor tunneling orbitals, respectively; H_(vμ) and S_(vμ) are coreHamiltonian and overlap matrix, and E₀ is a tunneling orbital energydefined by,

E₀ = ∑_(λ, ρ)D_(λ)F_(λρ)D_(ρ) = ∑_(λ, ρ)A_(λ)F_(λρ)A_(ρ)

where F_(μρ) is the reduced Fock matrix. The second equality in theabove equation corresponds to the resonance of the donor and acceptorenergies at the transition state of electron transfer reaction; inpractice, the resonance is achieved by applying a static electric fieldmimicking the action of the polar environment and solvation effects.

The equation below shows that the inter-atomic currents are primarilydetermined by the overlap of the tails of the two tunneling orbitals.The electronic Franck-Condon factor

$\left( {\prod\limits_{i}{s_{i}^{\alpha}{\prod\limits_{j \neq t}s_{j}^{\beta}}}} \right)$

contributes as a uniform scaling factor for all inter-atomic currents.The total atomic current through a given atom a,

${J_{a}^{tot} \equiv {\frac{1}{2}{\sum\limits_{b}{J_{a,b}}}}},$

is proportional to the probability that the tunneling electron ispassing through this atom; as such, it provides a convenient way ofidentifying atoms that constitute the tunneling pathways. The tunnelingmatrix element that determines the rate of Electron Transfer (ET) iscalculated as the total flux across the dividing surface between thedonor and acceptor redox complexes (the flux theorem; Stuchebrukhov(2001) Adv. Chem. Phys. 118:1),

$T_{DA} = {{- \hslash}{\sum\limits_{a \in S}{\sum\limits_{b \notin S}{J_{ab}.}}}}$

Here, the summation of interatomic tunneling currents between atoms ‘a’on one side, denoted as ‘S’ side, of the dividing surface and atoms ‘b’on the other side (Stuchebrukhov (1996) J. Chem. Phys. 104:8424). Theinter-atomic tunneling currents provide an internal assessment of thequality of calculation as a measure of conservation of the totaltunneling flux between the donor and acceptor redox centers. An exampleis shown in FIG. 6 for a model heme b_(L)→E heme b_(H) system with Phe90in the ON conformation. The flux is approximately conserved along thewhole tunneling pathway especially in the middle part, between donor andacceptor, as it should; to account for small variations, we compute thetunneling matrix element as an average over the middle region. All thestudied systems behave in a similar manner. The value of the tunnelingflux was taken as a measure of electron transfer rate in the analysis ofdifferent ligand-bound variants of bc₁ complex.

As displayed in FIG. 7, the binding of the 6 candidate ligands fromExample 1 at the NQ-site induces a series of conformational changes.Tyr95 undergoes moderate-to-substantial conformational changes thatinduce (along with a strong aromatic interaction due to the dockedligand) a conformational change of Phe91 residue, which ultimatelyattract the internal switch Phe90 residue from the intra-heme b regionto OFF position. In short, compared to the natural ON_(LH) conformation(which is attained by binding of QH₂ molecule at Q° site shown in FIG.7), upon binding of 6 candidate ligands at the NQ-site, Phe90 residueswings away by a torsion angle of 76.31° and clusters at a conformationwhich we designate as OFF_(NQ) (torsion angle equals 9.77° compared toON_(LH) with a torsion angle of 86.08°), as indicated in FIG. 7. Asshown in FIG. 8, in OFF_(NQ) conformation the electron tunneling flux isdramatically diminished, compared with the native ON_(LH) conformation,which is an indication of significantly reduced electron transfer ratebetween heme b_(L) and heme b_(H).

Example 2. Molecular Dynamic Simulations

Molecular dynamics (MD) simulations of the five discovered ligands(excluding the isomer ZINC17147426) bound to ligand-free bc₁ structure(PDB ID: 1BE3) were conducted in order to verify the binding affinitiesof those ligands to NQ-site and in addition to confirm the proposedinduced OFF_(NQ) conformational change of the internal switch Phe90residue. As shown in Table 2 the binding energies of the differentligands vary significantly in contrast to the results obtained above inthe docking simulations (Table 1). Ligands ZINC01691943 and ZINC01748097show the lowest stable binding energies while ligands ZINC01661542 andZINC17465983 show comparably higher binding energies, with ligandZINC17147424 having the highest binding energy of all.

TABLE 2 Binding energy averaged components for the five ligands atNQ-site during the MD simulation trajectory (in units of kcal/mol).E_(elec) E_(vdw) E_(polar) E_(SASA) E_(bind) ZINC01661542  −36.47(1.36)* 0.65 (0.25) 10.36 (0.88) −3.99 (0.11) −29.45 (0.83) ZINC01691943−30.52 (0.56) −56.19 (4.96) 17.91 (1.86) −3.38 (0.26) −72.17 (6.24)ZINC01748097 −35.57 (1.22) 2.44 (3.69) −9.16 (0.51)  −4.49 (0.23) −46.77(4.08) ZINC17147424 −21.22 (1.66) −8.70 (3.70) 14.22 (2.37) −2.81 (0.18)−18.50 (3.82) ZINC17465983 −29.44 (4.77) −3.38 (1.94) 13.34 (0.69) −3.57(0.16) −23.05 (6.17) *The values in parenthesis indicate the variationestimate of the corresponding quantity along the MD trajectory.

Analysis of the MD simulations trajectories for the different ligandsfurther validates their binding mode and proposed influence on Phe90conformation. As shown in Figure FIG. 9A, ligands ZINC01691943 andZINC01748097 remain bound to NQ-site during the whole 50 ns simulationtime and even get closer to heme b_(L)-hemeb_(H) system. Those twoligands show high magnitude of stability as indicated by their minimalRMSD during the simulation time (FIG. 9B). Nevertheless, only ligandZINC01691943 induces the desired conformational changes of Phe90 asindicated in FIG. 9C where the total computed distance of the displacedPhe90 is greater than that found in Q-bound bc₁ structure (PDB: 1NTZ,distance=13.3 Å) along the whole simulation time. Of the next twoligands ZINC01661542 and ZINC17465983, only ligand ZINC17465983 remainstightly-bound and produces the desired conformational changes of Phe90residue. Finally, as expected for having the highest binding energy,ligand ZINC17147424 undocks from NQ-site, shows the highest RMSD atNQ-site which decreases as it leaves the site and also fails to keep thedesired conformational changes of Phe90 residue.

Ultimately, ligand ZINC01691943 is the most promising one among the fiveligands while ligand ZINC17147424 is the least promising one.Visualization of MD snapshots of the two ligands along with the Phe90residue (and compare it to the other monomer Phe90 with a vacantQ_(o)/Q_(i)/NQ-sites) supports these conclusions. As shown in FIG. 10,ligand ZINC01691943 remains docked at NQ-site all over the simulationtime and even binds tighter over time (as indicated by draknessgradient). In addition, it displaces the Phe90 residue away from theintra-heme b region as compared to the other monomer Phe90 residue. Onthe other hand, ligand ZINC17147424 unbinds from NQ-site and leads toundesired conformational changes of Phe90 residue.

Example 3. Continued in Silico Screening of Ligand Binding Motifs

All currently-available 87,257 molecules was retrieved from the ZINCdatabase. The subset was processed further by AutoDockTools utility(Raccoon) to convert from Syby1 mol2 format to Autodock PDBQT format. Aseries of virtual screening calculations were carried out of the dockingof the ligands with the NQ-binding site using PyRx0.9.2 utilizing bothLamarckian Genetic Algorithm (LG) search method in Autodock 4.2 andAutodock Vina 4.2 advanced gradient optimization search method. Defaultsettings were used for both Autodock Vina and LG search methods. Toaccount for flexibility of the NQ-binding site and the differentpossible conformations of the Phe90 residue, we performed the dockingcalculation at both fixed and flexible residues (Phe90, Phe91, Ile92,Leu94 and Tyr95) modes as shown in FIG. 11.

From the data in FIG. 11, only 2,969 ligands show a preferential bindingby at least 1 kcal/mol at NQ-site in the flexible-residues docking modecompared to the fixed mode using Autodock Vina docking algorithm. Those2,969 ligands were challenged in a subsequent docking calculation atNQ-site using the Autodock Lamarckian Genetic (LG) algorithm as shown inFIG. 12. Only 353 ligands passed the second screening with at least 1kcal/mol in the flexible mode compared to the fixed mode. Additionallythose ligands were selected that have lower flexible-mode bindingaffinity to NQ-site compared to QH₂ molecule (which has binding energyfor NQ-site equals to −8.75 kcal/mol) in both docking algorithms.

In addition to the above criteria, we further restricted the ligandclass search such that the candidate ligands would not compete with QH₂molecule for binding at Q_(o) site. Further docking analysis of theresultant 353 ligands into the active Q_(o) site (as it exists in PDB1NTZ) reduced the number of ligands down to 41 candidates. As shown inFIG. 13, those 41 candidate ligands exhibit binding energy higher than−8.8 kcal/mol in both docking methods and hence would bindpreferentially at NQ-site, but not at Q_(o) site as compared to QH₂molecule.

Investigating the chemical structures of those 41 ligands reveals thatall share binding motifs composed of cyclic rings but differ in thenumber of fused, directly or indirectly-connected rings. Therefore weclassify those ligands under 4 different classes based on the number ofrings involved in the binding motifs and whether they are fused ordirectly/indirectly bonded as listed in Table 3 and shown in FIG. 14.

TABLE 3 Different binding motif classes of the NQ-site ligands and theircorresponding ligand IDs. Ligand Binding Class Motif Ligand ID I 2 fusedrings ZINC00045127, ZINC01323080, ZINC01677767, ZINC01701287,ZINC01706901, ZINC01758682 II 2 directly- or ZINC00627552, ZINC01681645,ZINC05518952 indirectly- bonded rings III 3 fused or ZINC01611570,ZINC01685490, ZINC01718184, bonded rings ZINC05464492, ZINC05699002,ZINC16889877, ZINC16968711 IV 4 or more fused ZINC01578373,ZINC03843643, ZINC03874978, or bonded rings ZINC03954046, ZINC04707030,ZINC06200106, ZINC08298762, ZINC16890026, ZINC16890132, ZINC16953369,ZINC16970002, ZINC16970006, ZINC16989825, ZINC17002577, ZINC17003067,ZINC17014023, ZINC17014031, ZINC01597160, ZINC01616623, ZINC01617881,ZINC01642065, ZINC01676502, ZINC01719516, ZINC05707314, ZINC17027383

In the molecular dynamics (MD) simulation study of Example 2, wediscovered that a ligand binding motif at NQ-SITE of two fused aromaticrings (such as the indole of ZINC01691943) can show lower binding energy(−72.17±6.24 kcal/mol) than that observed with a higher or lower numberof fused rings. In addition, we found that the conjugated aromaticity ofthe binding motif can contribute to lower binding energy (as seen withZINC17465983 compared to ZINC01661542, where both have binding motif ofthree fused rings while ZINC01661542 lacks the conjugated system).Finally, we believe that the aromatic character of the binding motif canimprove the switching off of the internal switch (LH, Phe90) througharomatic-aromatic interactions which induce the required conformationalchanges.

The Class I (such as ZINC00045127) ligands present natural extensions,in different directions, to the binding motif of two fused aromaticrings (benzene ring fused with pyrrole ring forming indole compound asshown in ligand ZINC01691943). The first extension is that the ligandsnot only furnish two binding motifs bonded covalently (as in ligandZINC17465983) or through a longer intervening chain (two covalent bonds,as in ligand ZINC01691943) but also they can be spiro-fused (share oneatom) as shown in ligand ZINC00045127. Moreover the ligandsZINC01323080, ZINC01677767, ZINC01701287, ZINC01706901 and ZINC01758682only have one binding motif. A further addition to the binding motifstructure is seen in those ligands where the fused ring system is not anindole one but rather a tetralin system (benzene ring fused withcyclohexane ring) or even larger heterocyclic ring (1,4 diazepine ring)fused with a benzene ring. Another finding regarding class I ligands isthat not all of them show a conjugated aromatic system, yet suchconjugated aromaticity can impart higher binding affinity and strongeraromatic-aromatic interaction inducing the desired conformationalchanges down to the internal switch Phe90 residue.

Class II ligands present a dramatic extension to class I ligands in thatclass II ligands show that the ligand binding motif can be composed oftwo covalently-bonded rings instead of two fused rings. Because in suchsystems the distance between the two ring edges is very close to that ofthree fused-rings system, the conjugated aromaticity can ensure properbinding and desired conformational induction. Otherwise, as weexperienced in the MD simulations of Example 1, the system lackingconjugated aromaticity (ZINC01661542) unbinds from the NQ-site muchearlier compared to the system with conjugated aromaticity(ZINC17465983).

Class III ligands can represent an upper limit of the ligand size(three-fused rings) that can fit in NQ-site and remain bound providedthat they have conjugated aromatic character. Based on the MDsimulations of Example 2, class IV ligands can be more prone to leak outof NQ-site.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

What is claimed is:
 1. A method of inhibiting respiratory complex III ina cell, comprising: contacting the cell with a compound having astructure selected from the group consisting of:

wherein each R¹, R², R³, R⁴, R⁵, and R⁶ is independently selected fromthe group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁₋₆haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl,oxide, —CN, —NH₂, —NO₂, and —C(O)—C₁₋₆ alkyl, and —C(O)O⁻; thesubscripts m and n are each independently integers from 0 to 5; thesubscript p is an integer from 0 to 4; and the subscripts q, u, and vare each independently integers from 0 to 2; such that the compoundbinds to respiratory complex III, thereby inhibiting respiratory complexIII.
 2. The method of claim 1, wherein the compound has the structure ofFormula III:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, each R³ is independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,and oxide; the subscripts m and n are each independently integers from 0to 5; and the subscript p is an integer from 0 to
 4. 3. The method ofclaim 1, wherein the compound has the structure of Formula IV:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, and the subscripts m and nare each independently integers from 0 to
 5. 4. The method of claim 1,wherein the compound has the structure of Formula V:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, each R³ is independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,and oxide; each R⁴ is independently selected from the group consistingof hydrogen and C₁₋₆ alkyl; the subscripts m and n are eachindependently integers from 0 to 5; the subscript p is an integer from 0to 4; and the subscript q is an integer from 0 to
 2. 5. The method ofclaim 1, wherein the compound has the structure of Formula VI:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, and the subscripts m and nare each independently integers from 0 to
 5. 6. The method of claim 1,wherein the compound has the structure of Formula VII:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, each R³ is independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,and oxide; the subscripts m and n are each independently integers from 0to 5; and the subscript p is an integer from 0 to
 4. 7. The method ofclaim 1, wherein the compound has the structure of Formula VIII:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, each R³ is independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,and oxide; each R⁴ is independently selected from the group consistingof hydrogen and C₁₋₆ alkyl; the subscripts m and n are eachindependently integers from 0 to 5; the subscript p is an integer from 0to 4; and the subscript q is an integer from 0 to
 2. 8. The method ofclaim 1, wherein the compound has the structure of Formula IX:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, each R³ is independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,and oxide; each R⁴ is independently selected from the group consistingof hydrogen and C₁₋₆ alkyl; the subscripts m and n are eachindependently integers from 0 to 5; the subscript p is an integer from 0to 4; and the subscript q is an integer from 0 to
 2. 9. The method ofclaim 1, wherein the compound has the structure of Formula X:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, each R³ is independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,and oxide; the subscripts m and n are each independently integers from 0to 5; and the subscript p is an integer from 0 to
 4. 10. The method ofclaim 1, wherein the compound has the structure of Formula XII:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, each R³ is independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,and oxide; each R⁴ is independently selected from the group consistingof hydrogen and C₁₋₆ alkyl; each R⁵ is independently selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, hydroxyl, and C(O)O⁻; thesubscripts m and n are each independently integers from 0 to 5; thesubscript p is an integer from 0 to 4; and the subscripts q and u areeach independently integers from 0 to
 2. 11. The method of claim 1,wherein the compound has the structure of Formula XIII:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, each R³ is independentlyselected from the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,and oxide; each R⁴ is independently selected from the group consistingof hydrogen and C₁₋₆ alkyl; each R⁵ is independently selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, hydroxyl, and C(O)O⁻; each R⁶is independently selected from the group consisting of hydrogen andhydroxyl; the subscripts m and n are each independently integers from 0to 5; the subscript p is an integer from 0 to 4; the subscripts q, u,and v are each independently integers from 0 to 2; and the subscript wis an integer from 0 to
 3. 12. The method of claim 1, wherein thecompound has the structure of Formula XIV:

wherein each R¹ is independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, halogen, hydroxyl, —NO₂, and—C(O)—C₁₋₆ alkyl; each R² is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₆₋₁₂ aryl, hydroxyl,—CN, —NH₂, —C(O)O—C₁₋₆ alkyl, and —C(O)O⁻, and the subscripts m and nare each independently integers from 0 to
 5. 13. The method of claim 1,wherein the compound is selected from group consisting of


14. The method of claim 1, wherein the cell is a cancer cell.
 15. Themethod of claim 14, wherein the cancer cell is in a subject.
 16. Themethod of claim 15, further comprising administering to the subject anamount of the compound effective to inhibit respiratory complex III inthe cancer cell.
 17. A method of treating cancer in a subject, themethod comprising administering to the subject in need thereof, atherapeutically effective amount of a compound having a structureselected from the group consisting of:

wherein each R¹, R², R³, R⁴, R⁵, and R⁶ is independently selected fromthe group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkylhydroxy, C₁₋₆haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₆₋₁₂ aryl, halogen, hydroxyl,oxide, —CN, —NH₂, —NO₂, and —C(O)—C₁₋₆ alkyl, and —C(O)O⁻; thesubscripts m and n are each independently integers from 0 to 5; thesubscript p is an integer from 0 to 4; the subscripts q, u, and v areeach independently integers from 0 to 2; and the subscript w is aninteger from 0 to 3.